This invention relates to novel potassium channels and genes encoding these channels. More specifically the invention provides isolated polynucleotides encoding the KCNQ4 potassium channel subunit, cells transformed with these polynucleotides, transgenic animals comprising genetic mutations, and the use of the transformed cells and the transgenic animals for the in vitro and in vivo screening of chemical compounds affecting KCNQ4 subunit containing potassium channels.
Potassium channels participate in the regulation of electrical signalling in excitable cells, and regulates the ionic composition of biological fluids. Mutations in the three known genes of the KCNQ branch of the K+-channel gene family underlie inherited cardiac arrhythmia""s, in some cases associated with deafness, and neonatal epilepsy.
Hearing loss is the most frequent sensory defect in humans. Hearing loss can be due to environmental and genetic factors, and the progressive hearing loss of the elderly (presbyacusis) most often seems to be due to a combination of both.
Inherited deafness can be classified as non-syndromic (isolated hearing loss) or syndromic (associated with other anomalies). Several hundred syndromes, consisting of hearing loss associated with defects in a variety of other organ systems, have been described. Non-syndromic deafness is classified according to its mode of inheritance as DFN, DFNA, and DFNB (X-linked, autosomal dominant and autosomal recessive, respectively). In general, autosomal recessive deafness has an early onset and is very severe. Autosomal dominant deafness, by contrast, more often develops slowly over several decades and may become apparent only in adulthood. It is hoped that genes identified in families with dominant deafness may alsoxe2x80x94with different types of mutationsxe2x80x94underlie some forms of presbyacusis.
A bewildering number of loci for non-syndromic deafness were identified in the last four years. There are at least 19 loci for autosomal dominant deafness (DFNA1 to DFNA19), and 22 loci for DFNB. Sometimes, depending on the particular mutation, the same gene can be involved in dominant or recessive deafness. This large number of loci reflects the complexity of the inner ear. Identification of these genes and characterisation of their products will significantly advance our understanding of the molecular basis of the physiology of this sensory organ.
Several genes involved in syndromic and non-syndromic deafness have already been identified and are reviewed by Petit [Petit C: Genes responsible for human hereditary deafness: symphony of a thousand; Nature Genet. 1996 14 385-391] and Kalatzis and Petit [Kalatzis V and Petit C: The fundamental and medical impacts of recent progress in research on hereditary hearing loss; Hum. Mol. Genet. 1998 7 1589-1597]. Among others, their gene products include transcription factors, unconventional myosin isoforms, xcex1-tectorin (an extracellular matrix protein), diaphanous, a protein interacting with the cytoskeleton, connexin 26 (a gap junction protein), and two genes encoding potassium channel subunits, KCNQ1 and KCNE1.
Ion channels play important roles in signal transduction and in the regulation of the ionic composition of intra- and extracellular fluids. Mutations in ion channels were since long suspected as possibly underlying some forms of hearing loss. In the cochlea (the auditory sensory organ), the transduction current through the sensory cells is carried by potassium ions and depends on the high concentration of that ion in the endolymph. So far only two genes encoding potassium channel subunits, KCNQ1 and KCNE1, were found to be mutated in syndromic hereditary deafness. The gene products of both genes, the KCNQ1 (or KvLQT1) and the mink (or IsK) protein, respectively, form heteromeric potassium channels.
KCNQ1 is a typical member of the voltage-gated potassium channel superfamily with 6 transmembrane domains and a pore region situated between the fifth and the sixth transmembrane domain. The minK protein has a single transmembrane span and cannot form potassium channels on its own. However, as a xcex2-subunit it enhances and modifies currents mediated by KCNQ1. These heteromeric channels participate in the repolarization of the heart action potential. Certain mutations in either KCNQ1 or KCNE1 cause a form of the autosomal dominant long QT syndrome (LQTS), a disease characterised by repolarization anomalies of cardiac action potentials resulting in arrhythmias and sudden death. Interestingly, other mutations in either gene lead to the recessive Jervell and Lange-Nielsen (JLN) syndrome that combines LQTS with congenital deafness. In order to cause deafness, KCNQ1/minK currents must be reduced below levels that are already sufficiently low to cause cardiac arrhythmia.
We have now cloned and characterised KCNQ4, a novel member of the KCNQ family of potassium channel proteins. KCNQ4 has been mapped to the DFNA2 locus for autosomal dominant hearing loss, and a dominant negative KCNQ4 mutation that causes deafness in a DFNA2 pedigree was identified.
KCNQ4 is the first potassium channel gene underlying non-syndromic deafness. KCNQ4 forms heteromeric channels with other KCNQ channel subunits, in particular KCNQ3.
The present invention has important implications for the characterisation and exploitation of this interesting branch of the potassium channel super family, as well as for the understanding of the cochlear physiology, and for human deafness and progressive hearing loss.
Accordingly, in its first aspect, the invention provides an isolated polynucleotide having a nucleic acid sequence which is capable of hybridising under high stringency conditions with the polynucleotide sequence presented as SEQ ID NO: 1, its complementary strand, or a sub-sequence thereof.
In another aspect the invention provides a recombinantly produced polypeptide encoded by the .polynucleotide of the invention.
In a third aspect the invention provides a cell genetically manipulated by the incorporation of a heterologous polynucleotide of the invention.
In a fourth aspect the invention provides a method of screening a chemical compound for inhibiting or activating or otherwise modulating the activity on a potassium channel comprising at least one KCNQ4 channel subunit, which method comprises the steps of subjecting a KCNQ4 channel subunit containing cell to the action of the chemical compound; and monitoring the membrane potential, the current, the potassium flux, or the secondary calcium influx of the KCNQ4 channel subunit containing cell.
In a fifth aspect the invention relates to the use of a polynucleotide sequence of the invention for the screening of genetic materials from humans suffering from loss of hearing (e.g. dominant, recessive, or otherwise), tinnitus, and other neurological diseases for mutations in the KCNQ4 gene.
In a sixth aspect the invention relates to the chemical compound identified by the method of the invention, in particular to the use of such compounds for diagnosis, treatment or alleviation of a disease related to tinnitus; loss of hearing, in particular progressive hearing loss, neonatal deafness, and presbyacusis (deafness of the elderly); and diseases or adverse conditions of the CNS, including affective disorders, Alzheimer""s disease, anxiety, ataxia, CNS damage caused by trauma, stroke or neurodegenerative illness, cognitive deficits, compulsive behaviour, dementia, depression, Huntington""s disease, mania, memory impairment, memory disorders, memory dysfunction, motion disorders, motor disorders, neurodegenerative diseases, Parkinson""s disease and Parkinson-like motor disorders, phobias, Pick""s disease, psychosis, schizophrenia, spinal cord damage, stroke, and tremor.
In a seventh aspect the invention provides a transgenic animal comprising a knock-out mutation of the endogenous KCNQ4 gene, a replacement by or an additional expression of a mutated KCNQ4 gene, or genetically manipulated in order to over-express the KCNQ4 gene or to over-express mutated KCNQ4 gene.
In an eighth aspect the invention relates to the use of the transgenic animal of the invention for the in vivo screening of therapeutic compounds.
Other objects of the invention will be apparent to the person skilled in the art from the following detailed description and examples.
The present invention provides novel voltage-gated potassium channels and genes encoding these channels. The invention also provides cells transformed with these genes, transgenic animals comprising genetic mutations, and the use of the transformed cells and the transgenic animals for the in vitro and in vivo screening of drugs affecting KCNQ4 containing potassium channels.
Polynucleotides
In its first aspect, the invention provides novel polynucleotides.
The polynucleotides of the invention are such which have a nucleic acid sequence capable of hybridising under high stringency conditions with the polynucleotide sequence presented as SEQ ID NO: 1, its complementary strand, or a sub-sequence thereof.
The polynucleotides of the invention include DNA, cDNA and RNA sequences, as well as anti-sense sequences, and include naturally occurring, synthetic, and intentionally manipulated polynucleotides. The polynucleotides of the invention also include sequences that are degenerate as a result of the genetic code.
As defined herein, the term xe2x80x9cpolynucleotidexe2x80x9d refers to a polymeric form of nucleotides of at least 10 bases in length, preferably at least 15 bases in length. By xe2x80x9cisolated polynucleotidexe2x80x9d is meant a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5xe2x80x2 end and one on the 3xe2x80x2 end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes recombinant DNA which is incorporated into an expression vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule, e.g. a cDNA, independent from other sequences.
The polynucleotides of the invention also include allelic variants and xe2x80x9cmutated polynucleotidesxe2x80x9d comprising a nucleotide sequence that differs from the sequence presented as SEQ ID NO: 1 at one or more nucleotide positions. The mutated polynucleotide may in particular be a polynucleotide of the invention comprising a nucleotide sequence as in SEQ ID NO: 1, which sequence, however, differs from SEQ ID NO: 1 so as to effect the expression of a variant polypeptide. The mutated polynucleotide may be a polynucleotide of the invention having a nucleotide sequence encoding a potassium channel having an amino acid sequence that has been changed at one or more positions. The mutated polynucleotide may in particular be a polynucleotide of the invention having a nucleotide sequence encoding a potassium channel having an amino acid sequence that has been changed at one or more positions located in the conserved regions, as defined by Table 1, below.
In a more specific embodiment the polynucleotide of the invention has the polynucleotide sequence giving rise to the G285S mutation as indicated in SEQ ID NO: 1, i.e. the DNA sequence that at position 935-937 holds the codon AGC rather than the codon GGC stated in SEQ ID NO: 1.
Hybridisation Protocol
The polynucleotides of the invention are such which have a nucleic acid sequence capable of hybridising with the polynucleotide sequence presented as SEQ ID NO: 1, its complementary strand, or a sub-sequence thereof, under at least medium, medium/high, or high stringency conditions, as described in more detail below.
In a preferred embodiment the polynucleotide is a fragment of at least 15 bases in length which is sufficient to permit the fragment to hybridise to DNA that encodes a polypeptide of the invention, preferably the polypeptide comprising the amino acid sequence presented as SEQ ID NO: 2 under at least medium, medium/high, or high stringency conditions, as described in more detail below.
Suitable experimental conditions for determining hybridisation between a nucleotide probe and a homologous DNA or RNA sequence, involves pre-soaking of the filter containing the DNA fragments or RNA to hybridise in 5xc3x97SSC [Sodium chloride/Sodium citrate; cf. Sambrook et al.; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. 1989] for 10 minutes, and pre-hybridisation of the filter in a solution of 5xc3x97SSC, 5xc3x97Denhardt""s solution [cf. Sambrook et al.; Op cit.], 0.5% SDS and 100 xcexcg/ml of denatured sonicated salmon sperm DNA [cf. Sambrook et al.; Op cit.], followed by hybridisation in the same solution containing a concentration of 10 ng/ml of a random-primed [Feinberg A P and Vogelstein B; Anal. Biochem. 1983 132 6-13], 32PdCTP-labeled (specific activity greater than 1xc3x97109 cpm/xcexcg) probe for 12 hours at approximately 45xc2x0 C. The filter is then washed twice for 30 minutes in 2xc3x97SSC, 0.5% SDS at a temperature of at least at least 60xc2x0 C. (medium stringency conditions), preferably of at least 65xc2x0 C. (medium/high stringency conditions), more preferred of at least 70xc2x0 C. (high stringency conditions), and even more preferred of at least 75xc2x0 C. (very high stringency conditions).
Molecules to which the oligonucleotide probe hybridises under these conditions may be detected using a x-ray film.
DNA Sequence Homology
In a preferred embodiment, the polynucleotides of the invention show a homology of at least 50%, preferably at least70%, more preferred at least 80%, even more preferred at least 90%, most preferred at least 95%, with the polynucleotide sequence presented as SEQ ID NO: 1.
As defined herein, the DNA sequence homology may be determined as the degree of identity between two DNA sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package [Needleman S B and Wunsch C D, Journal of Molecular Biology 1970 48 443-453] using default para meters suggested herein.
Cloned Polynucleotides
The isolated polynucleotide of the invention may in particular be a cloned polynucleotide.
As defined herein, the term xe2x80x9ccloned polynucleotidexe2x80x9d, refers to a polynucleotide or DNA sequence cloned in accordance with standard cloning procedures currently used in genetic engineering to relocate a segment of DNA, which may in particular be cDNA, i.e. enzymatically derived from RNA, from its natural location to a different site where it will be reproduced.
Cloning may be accomplished by excision and isolation of the desired DNA segment, insertion of the piece of DNA into the vector molecule and incorporation of the recombinant vector into a cell where multiple copies or clones of the DNA segment will be replicated, by reverse transcription of mRNA (reverse transcriptase technology), and by use of sequence-specific oligonucleotides and DNA polymerase in a polymerase chain reaction (PCR technology).
The cloned polynucleotide of the invention may alternatively be termed xe2x80x9cDNA constructxe2x80x9d or xe2x80x9cisolated DNA sequencexe2x80x9d, and may in particular be a complementary DNA (cDNA).
It is well established that potassium channels may be formed as heteromeric channels, composed of different subunits. Also it has been found that the potassium channel of the invention may form heteromers with other KCNQ""s, in particular KCNQ3. when co-expressed with these subunits. In addition, potassium channels can also associate with non-homologous subunits, e.g. the KCNE1 (formerly known as minK) subunit, that can functionally modulate these channels or lead to a specific localisation within the cell.
Therefore, in a preferred embodiment, the polynucleotide of the invention is cloned and either expressed by itself or co-expressed with polynucleotides encoding other subunits, in particular a polynucleotide encoding a KCNQ3 channel subunit.
Biological Sources
The isolated polynucleotide of the invention may be obtained from any suitable source. In a preferred embodiment, which the polynucleotide of the invention is cloned from, or produced, on the basis of a cDNA library, e.g. of the retina, brain, skeletal muscle. Commercial cDNA libraries are available from e.g. Stratagene and Clontech.
The isolated polynucleotide of the invention may be obtained by methods known in the art, e.g. those described in the working examples below.
In a preferred embodiment the polynucleotide of the invention may be obtained using the PCR primers described in the working examples and presented as SEQ ID NOS: 3-32.
Preferred Polynucleotides
In a preferred embodiment, polynucleotide of the invention comprises the polynucleotide sequence presented as SEQ ID NO: 1.
In another preferred embodiment the polynucleotide of the invention is a sequence giving rise to KCNQ4 channels subunits comprising one or more substitutions.
In another preferred embodiment the polynucleotide of the invention is a sequence giving rise to KCNQ4 channels subunits comprising one or more substitutions in the conserved regions, as defined in more details below.
In a more preferred embodiment the polynucleotide of the invention has a polynucleotide sequence giving rise to the G285S mutation as indicated in SEQ ID NO: 1, e.g. the DNA sequence that at position 935-937 holds the codon AGC rather than the codon GGC stated in SEQ ID NO: 1.
Also contemplated within the scope of this invention are the primer sequences used in Example 2 below for the amplification of the single KCNQ4 exons, that can then be screened for mutations.
Therefore, in another preferred embodiment the polynucleotide of the invention is a primer sequence comprising any one of the polynucleotide sequences presented as SEQ ID NOS: 3-32.
It has been demonstrated that KCNQ channels often show alternative splicing and therefore may occur as isoforms originating from the same gene. Such isoforms as well as the different cDNA sequences from which they occurred are also contemplated within the scope of the present invention.
Finally the genes encoding KCNQ channel subunits in other species have been found to differ slightly from the human genes. However, genes of other species, e.g. mouse, rat, monkey, rabbit, etc., are also contemplated within the scope of the present invention.
Recombinantly Produced Polypeptides
In another aspect the invention relates to substantially pure functional polypeptides that have the electrophysiological and pharmacological properties of a KCNQ4 channel, or KCNQ4 channel subunits. The novel polypeptides of the invention may be obtained by the polynucleotides of the invention using standard recombinant DNA technology.
In a preferred embodiment, a polypeptide of the invention is the KCNQ4 potassium channel subunit comprising the amino acid sequence presented as SEQ ID NO: 2, and biologically active fragments hereof.
Modifications of this primary amino acid sequence may result in proteins which have substantially equivalent activity as compared to the unmodified counterpart polypeptide, and thus may be considered functional analogous of the parent proteins. Such modifications may be deliberate, e.g. as by site-directed mutagenesis, or they may occur spontaneous, and include splice variants, isoforms, homologues from other species, and polymorphisms, and include the variant KCNQ4/G285S, that is described in more detail below. Such functional analogous are also contemplated according to the invention.
Moreover, modifications of this primary amino acid sequence may result in proteins which do not retain the biological activity of the parent protein, including dominant negative forms, etc. A dominant negative protein may interfere with the wild-type protein by binding to, or otherwise sequestering regulating agents, such as upstream or downstream components, that normally interact functionally with the polypeptide. Such dominant negative forms are also contemplated according to the invention.
In the context of this invention, the term xe2x80x9cvariant polypeptidexe2x80x9d means a polypeptide (or protein) having an amino acid sequence that differs from the sequence presented as SEQ ID NO; 2 at one or more amino acid positions. Such variant polypeptides include the modified polypeptides described above, as well as conservative substitutions, splice variants, isoforms, homologues from other species, and polymorphisms, and includes the variant KCNQ4/G285S (i.e. KCNQ4/G333S according to the KCNQ1 numbering).
As defined herein, the term xe2x80x9cconservative substitutionsxe2x80x9d denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as, the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like. The term conservative substitution also include the use of a substituted amino acid residue in place of an un-substituted parent amino acid residue provided that antibodies raised to the substituted polypeptide also immunoreact with the un-substituted polypeptide.
Also contemplated within the scope of this invention are the oligopeptides encoded by the primer sequences used in Example 2 below for the amplification of the single KCNQ4 exons, that can then be screened for mutations.
KCNQ1 Numbering System
In the context of this invention, amino acid residues (as well as nucleic acid bases) are specified using the established one-letter symbol.
By aligning the amino acid sequences of a polypeptide of the present invention to those of the known polypeptides, a specific amino acid numbering system may be employed, by which,system it is possible to unambiguously allot an amino acid position number to any amino acid residue in any KNCQ channel protein, which amino acid sequence is known.
Such an alignment is presented in Table 1, below. Using the ClustalX computer alignment program [Thompson J D, Gibson T J, Plewniak F, Jeanmougin F, and Higgins D G: The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools; Nucleic Acids Res. 1997 25 (24) 4876-82], and the default parameters suggested herein, the amino acid sequence of a polypeptide of the present invention (hKCNQ4) and the amino acid sequences of the known polypeptides hKCNQ2-3 are; aligned with, and relative to, the amino acid sequences of the known polypeptide hKCNQ1 (formerly known as KvLQT1). In the context of this invention this numbering system is designated the KCNQ1 Numbering System.
In describing the various protein variants produced or contemplated according to the invention, the following nomenclatures have been adapted for ease of reference:
According to this nomenclature the substitution of serine for glycine at position 333 is designated as xe2x80x9cG333Sxe2x80x9d.
A deletion of glycine at the same position is designated xe2x80x9cG333*xe2x80x9d.
An insertion of an additional amino acid residue, in this example lysine, may be designated xe2x80x9cG333GKxe2x80x9d or xe2x80x9c*334Kxe2x80x9d (assumed that no position exists for this position in the amino acid sequence used for establishing the numbering system).
An insertion of an amino acid residue, in this example valine, at a position which exists in the established numbering system, but where no amino acid residue is actually present, may be designated xe2x80x9cxe2x88x92301Vxe2x80x9d.
Biological Activity
The polynucleotide of the invention encodes a potassium channel subunit, which has been termed KNCQ4. In the cochlea, it is differentially expressed in sensory outer hair cells. A mutation in this gene in a pedigree with autosomal dominant hearing loss changes a residue in the KCNQ4 pore region. It abolishes the outwardly rectifying potassium currents of wild-type KCNQ4 on which it exerts a strong dominant negative effect.
Ion channels are excellent targets for drugs. KCNQ4, or heteromeric channels containing the KCNQ4 subunit, may be a particularly interesting target for the treatment of tinnitus and the prevention or treatment of progressive hearing loss.
KCNQ Channels in Genetic Disease
It is remarkable that mutations in every known KCNQ gene lead to human disease: Mutations in KCNQ1 (KvLQT1) cause the autosomal dominant long QT syndrome (LQTS), and, when present on both alleles, the Jervell and Lange-Nielsen (JLN) syndrome whose symptoms include deafness in addition to cardiac arrhythmias. Mutations in either KCNQ2 or KCNQ3, which form heteromers that probably represent the M-channel, cause benign familial neonatal convulsions (BFNC). The present invention adds KCNQ4 and the associated autosomal dominant deafness to that list.
After KCNQ1, KCNQ4 is now the second KCNQ channel whose loss of function leads to deafness.
Therefore, in a preferred embodiment of the invention, mutated polynucleotides may be employed in the screening for drugs that affect diseases associated with such mutations in the KCNQ4 gene.
Heteromers Formed by KCNQ Subunits
The KCNQ channels described so far function physiologically as heteromers. KCNQ1 associates with KCNE1 (formerly known as minK), and KCNQ2 and KCNQ3 form heteromeric channels that underlie the M-current, an important determinant of neuronal excitability that is regulated by several neurotransmitters.
Like other KCNQ channel subunits, KCNQ4 may interact with other subunits, e.g. KCNE1 or other KCNQ channel subunits, and in particular with KCNQ3. Currents from homomeric KCNQ3 are very small and often cannot be distinguished from Xenopus oocyte background currents. Co-expression of KCNQ3 with KCNQ4 markedly increased current amplitudes. Significantly, heteromeric KCNQ3/KCNQ4 channels activated faster than homomeric KCNQ4 channels, the voltage-dependence was shifted to more negative potentials, and currents displayed a different drug sensitivity.
Antibodies
The polypeptides of the invention can be used to produce antibodies which are immunoreactive or bind to epitopes of these polypeptides. Antibodies which consist essentially of pooled monoclonal antibodies with different specificities, as well as distinct monoclonal antibody preparations may be provided.
The preparation of polyclonal and monoclonal antibodies is well known in the art. Polyclonal antibodies may in particular be obtained as described by e.g. Green et al.: xe2x80x9cProduction of Polyclonal Antiseraxe2x80x9d in Immunochemical Protocols (Manson, Ed.); Humana Press, 1992, Pages 1-5; and Coligan et al.: xe2x80x9cProduction of Polyclonal Antisera in rabbits, rats, Mice and Hamstersxe2x80x9d in Current Protocols in Immunology, 1992, Section 2.4.1; which protocols are hereby incorporated by reference.
Monoclonal antibodies may in particular be obtained as described by e.g. Kohler and Milstein, Nature 1975 256 495; Coligan et al. in Current Protocols in Immunology, 1992, Sections 2.5.1-2.6.7; and Harlow et al. in Antibodies: A Laboratory Manual; Cold Spring Harbor Pub., 1988, Page 726; which protocols are hereby incorporated by reference.
Briefly, monoclonal antibodies may be obtained by injecting e.g. mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce the antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques, including affinity chromatography with protein A Sepharose, size-exclusion chromatography, and ion-exchange chromatography, see. e.g. Coligan et al. in Current Protocols in Immunology, 1992, Sections 2.7.1-2.7.12, and Sections 2.9.1-2.9.3; and Barnes et al.: xe2x80x9cPurification of Immunoglobulin G (IgG)xe2x80x9d in Methods in Molecular Biology; Humana Press, 1992, Vol. 10, Pages 79-104.
The polyclonal or monoclonal antibodies may optionally be further purified, e.g. by binding to and elution from a matrix to which the polypeptide, to which the antibodies were raised, is bound.
Antibodies which bind to the polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunising antigen. The polypeptide used to immunise an animal may be obtained by recombinant DNA techniques or by chemical synthesis, and may optionally be conjugated to a carrier protein. Commonly used carrier proteins which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide may then be used to immunise the animal, which may in particular be a mouse, a rat, a hamster or a rabbit.
Genetically Manipulated Cells
In a third aspect the invention provides a cell genetically manipulated by the incorporation of the heterologous polynucleotide of the invention. The cell of the invention may in particular be genetically manipulated to transiently or stably express, over-express or co-express a KCNQ4 channel subunit as defined above. Methods of transient and stable transfer are known in the art.
The polynucleotide of the invention may be inserted into an expression vector, e.g. a plasmid, virus or other expression vehicle, and operatively linked to expression control sequences by ligation in a way that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. Suitable expression control sequences include promoters, enhancers, transcription terminators, start codons, splicing signals for introns, and stop codons, all maintained in the correct reading frame of the polynucleotide of the invention so as to permit proper translation of mRNA. Expression control sequences may also include additional components such as leader sequences and fusion partner sequences.
The promoter may in particular be a constitutive or an inducible promoter. When cloning in bacterial systems, inducible promoters such as pL of bacteriophage xcex3, plac, ptrp, ptac (ptrp-lac hybrid promoter), may be used. When cloning in mammalian systems, promoters derived from the genome of mammalian cells, e.g. the TK promoter or the metallothionein promoter, or from mammalian viruses, e.g. the retrovirus long terminal repeat, the adenovirus late promoter or the vaccinia virus 7.5K promoter, may be used. Promoters obtained by recombinant DNA or synthetic techniques may also be used to provide for transcription of the polynucleotide of the invention.
Suitable expression vectors typically comprise an origin of expression, a promoter as well as specific genes which allow for phenotypic selection of the transformed cells, and include vectors like the T7-based expression vector for expression in bacteria [Rosenberg et al; Gene 1987 56 125], the pMSXND expression vector for expression in mammalian cells [Lee and Nathans, J. Biol. Chem. 1988 263 3521], baculovirus derived vectors for expression in insect cells, and the oocyte expression vector PTLN [Lorenz C, Pusch M and Jentsch T J: Heteromultimeric CLC chloride channels with novel properties; Proc. Natl. Acad. Sci. USA 1996 93 13362-13366].
In a preferred embodiment, the cell of the invention is an eukaryotic cell, in particular a mammalian cell, an oocyte, or a yeast cell. In a more preferred embodiment, the cell of the invention is a human embryonic kidney (HEK) cell, a HEK 293 cell, a BHK21 cell, a Chinese hamster ovary (CHO) cell, a Xenopus laevis oocyte (XLO) cell, a COS cell, or, any other cell line able to express KCNQ potassium channels.
When the cell of the invention is an eukaryotic cell, incorporation of the heterologous polynucleotide of the invention may be in particular be carried out by infection (employing a virus vector), by transfection (employing a plasmid vector), or by calcium phosphate precipitation, microinjection, electroporation, lipofection, or other physical-chemical methods known in the art.
In a further preferred embodiment, the cell of the invention is genetically manipulated to co-express KCNQ4 and KCNQ1 channel subunits; KCNQ4 and KCNQ2 channel subunits; KCNQ4 and KCNQ3 channel subunits; KCNQ4 and KCNQ1 and KCNQ2 channel subunits; KCNQ4 and KCNQ1 and KCNQ3 channel subunits; KCNQ4 and KCNQ2 and KCNQ3 channel subunits; or KCNQ4 and KCNO1 and KCNQ2 and KCNQ3 channel subunits.
KCNQ4 Active Chemical Compounds
In another aspect the invention relates to chemical compounds capable of binding to, and showing activity at potassium channels containing one or more KCNQ4 subunits. In the context of this invention such compounds are termed KCNQ4 active compounds. The KCNQ4 active compounds of the invention show activity in concentrations below 100 xcexcM, preferably below 10 xcexcM, more preferred below 1 xcexcm. In its most preferred embodiment the KCNQ4 active compounds of the invention show activity in low micromolar and the nanomolar range.
The KCNQ4 active compounds of the invention have therapeutic potential, and may be used for the manufacture of pharmaceutical compositions.
The KCNQ4 active compounds of the invention may in particular be used in diagnosis, treatment, prevention or alleviation of diseases related to tinnitus, loss of hearing, in particular progressive hearing loss, neonatal deafness, and presbyacusis (deafness of the elderly); and diseases or adverse conditions of the CNS, including affective disorders, Alzheimer""s disease, anxiety, ataxia, CNS damage caused by trauma, stroke or neurodegenerative illness, cognitive deficits, compulsive behaviour, dementia, depression, Huntington""s disease, mania, memory impairment, memory disorders, memory dysfunction, motion disorders, motor disorders, neurodegenerative diseases, Parkinson""s disease and Parkinson-like motor disorders, phobias, Pick""s disease, psychosis, schizophrenia, spinal cord damage, stroke, and tremor.
Currently two compound have been identified. As a preferred embodiment the invention therefore provides 1,3-dihydro-1-phenyl-3,3-bis(4-pyridylmethyl)-2H-indol-2-one (Linopirdine) and 10,10-bis(4-pyridinyl-methyl)-9(10H)-antracenone (XE991) for use in the manufacture of a pharmaceutical composition for the diagnosis, treatment, prevention or alleviation of the above diseases.
Screening of Drugs
In a further aspect the invention provides methods for screening for KCNQ4 active compounds, i.e. chemical compounds capable of binding to, and showing activity at potassium channels containing one or more KCNQ4 subunits. The activity determined may be inhibitory activity, stimulating activity, or other modulatory activity. In particular the KCNQ4 active compound may induce a second messenger response, which cause a change of the molecular characteristics of the cell, e.g. the ion flux, enzyme activation, changes in cyclic nucleotides such as cAMP, cADP, cGMP, and cGDP, etc.
Therefore, in another aspect, the invention provides a method for identifying functional ligands for a human potassium channel, comprising a KCNQ4 subunit, which method comprises transfecting cells with one or more polypeptides of the invention, encoding a KCNQ4 channel subunit, and detecting the effect on the signal transduction pathway caused in these cells by binding of the ligands to the receptor by a reporter system.
Such chemical compounds can be identified by one of, or both methods described below.
Binding Studies
Binding studies are usually carried out by subjecting the target to binding with a labelled, selective agonist (binding agent), to form a labelled complex, followed by determination of the degree of displacement caused by the test compound upon addition to the complex.
In a specific aspect the invention provides a method of screening a chemical compound for capability of binding to a potassium channel comprising at least one KCNQ4 channel subunit, which method comprises the steps of (i) subjecting a KCNQ4 channel subunit containing cell to the action of a KCNQ4 binding agent to form a complex with the KCNQ4 channel subunit containing cell; (ii) subjecting the complex of step (i) to the action of the chemical compound to be tested; and (iii) detecting the displacement of the KCNQ4 binding agent from the complex with the KCNQ4 channel subunit containing cell.
The KCNQ4 channel subunit containing cell preferably is a cell of the invention as described above.
The KCNQ4 binding agent preferably is a radioactively labelled 1,3-dihydro-1-phenyl-3,3-bis(4-pyridylmethyl)-2H-indol-2-one (Linopirdine); or 10,10-bis(4-pyridinyl-methyl)-9(10H)-antracenone.
In a even more preferred embodiment, the biding agent is labelled with 3H, and the displacement of the KCNQ4 binding agent from the complex with the KCNQ4 channel subunit containing cell is detected by measuring the amount of radioactivity by conventional liquid scintillation counting.
Activity Studies
The KCNQ4 channel agonists may affect the potassium channel in various ways. The agonist may in particular show inhibitory activity, stimulating activity, or other modulatory activity.
In a specific aspect the invention provides a method for determining the activity at potassium channels containing one or more KCNQ4 subunits. According to this method a KCNQ4 channel subunit containing cell is subjecting to the action of the chemical compound to be tested, and the activity is detected by way of monitoring the membrane potential, the current, the potassium flux, or the secondary calcium influx of the KCNQ4 channel subunit containing cell, preferably a genetically manipulated as described above.
The membrane potential and the current may be monitored by electrophysiologic methods, including patch clamp techniques, such as current clamp technology and two-electrode voltage clamp technology, or by spectroscopic methods, such as fluorescence methods.
In a preferred embodiment, monitoring of the membrane potential of the KCNQ4 channel subunit containing cell is performed by patch clamp techniques.
In another preferred embodiment, monitoring of the membrane potential of the KCNQ4 channel subunit containing cell is performed by spectroscopic methods, e.g. using fluorescence methods. In a more specific embodiment, the KCNQ4 channel subunit containing cell is mixed with a membrane potential indicating agent, that allow for a determination of changes in the membrane potential of the cell, caused by the addition of the test compound. The membrane potential indicating agent may in particular be a fluorescent indicator, preferably DIBAC4(3), DiOC5(3), and DiOC2(3).
In yet a preferred embodiment, monitoring of the membrane potential of the KCNQ4 channel subunit containing cell is performed by spectroscopic methods, e.g. using a FLIPR assay (Fluorescence Image Plate Reader; available from Molecular Devices).
Screening of Genetic Material
In a further aspect the invention relates to the use of a polynucleotide sequence of the invention for the screening of genetic materials. By this method, individuals bearing a gene identical or homologous to a polynucleotide of the invention may be identified.
In the screening method of the invention, a polynucleotide of the invention, or any fragment or sub-sequence hereof, and in particular any one of the polynucleotide sequences presented as SEQ ID NOS: 3-32, is employed. For the identification of individuals bearing mutated genes, preferably a mutated form of the polynucleotide represented by SEQ ID NO: 1 is employed, and in particular a polynucleotide sequence holding the mutation giving rise to the KCNQ4/G285S variant.
In the screening method of the invention only short sequences needs to be employed depending on the actual method used. For SSCA, several hundreds of base pairs may be needed, for oligonucleotide or PCR hybridisation only of from about 10 to about 50 basepairs may be needed.
In a more specific embodiment, the primer sequences used in Example 2 below for the amplification of the single KCNQ4 exons, and presented as SEQ ID NOS: 3-32, may be used for the screening of mutations.
The screening may be accomplished by conventional methods, including hybridisation, SSCA analysis, and array technology (DNA chip technology). The hybridisation protocol described above represents a suitable protocol for use in a screening method of the invention.
Therefore, in particular embodiment the invention provides a method for the identification, localisation, isolation or amplification a polynucleotide of the invention, which method a polynucleotide primer of the invention, in particular any one of those presented as SEQ ID NOS: 3-32, is used as a probe. This method may be accomplished using conventional molecular biological techniques, e.g. those used and described in the working examples below.
In a preferred embodiment, the method is used for performing gene amplification using conventional PCR techniques, e.g. as described in the working examples below.
Transgenic Animals
Transgenic animal models provide the means, in vivo, to screen for therapeutic compounds. The establishment of transgenic animals may in particular be helpful for the screening of drugs to fully elucidate the pathophysiology of KCNQ4/DFNA2 deafness. These animals may also be valuable as a model for the frequent condition of presbyacusis that also develops slowly over decades. Since KCNQ4 is expressed also in brain, they may also be helpful in screening for drugs effective in CNS disorders, e.g. epilepsy.
By transgene is meant any piece of polynucleotide which is inserted by artifice into a cell, and thus becomes part of the genome of the organism that develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e. foreign) to the transgenic organism, or it may represent a gene homologous to an endogenous gene of the organism.
By a transgenic animal is meant any organism holding a cell which includes a polynucleotide sequence which is inserted into that cell by artifice, and which cell becomes part of the transgenic organism which develops from that cell. Such a transgene may be partly or entirely heterologous to the transgenic animal. Although transgenic mice represent a preferred embodiment of the invention, other transgenic mammals including, but not limited to transgenic rodents (e.g. hamsters, guinea pigs, rabbits and rats), and transgenic pigs, cattle, sheep and goats may be created by standard techniques and are included in the invention.
Preferably, the transgene is inserted by artifice into the nuclear genome.
Knock-out and Knock-in Animals
The transgenic knock-out animal models may be developed by homologous recombination of embryonic stem cells with constructs containing genomic sequence from the KCNQ4 gene, that lead to a loss of function of the gene. after insertion into the endogenous gene.
By knock-out mutation is meant an alteration in the polynucleotide sequence that reduces the biological activity of the polypeptide normally encoded therefrom. In order to create a true knock-out model, the biological activity of the expressed polypeptide should be reduced by at least 80% relative to the un-mutated gene. The mutation may in particular be a substitution, an insertion, a deletion, a frameshift mutation, or a mis-sense mutation. Preferably the mutation is a substitution, an insertion or a deletion.
To further assess the role of KCNQ4 at an organism level, the generation of an animal, preferably a mouse, lacking the intact KCNQ4 gene, or bearing a mutated KCNQ4 gene, is desired.
A replacement-type targeting vector, which may be used to create a knock-out model, may be constructed using an isogenic genomic clone, e.g. from a mouse strain such as 129/Sv (Stratagene Inc., La Jolla, Calif.). The targeting vector may be introduced into a suitably-derived line of embryonic stem (ES) cells by electroporation to generate ES cell lines that carry a profoundly truncated form of the KCNQ4 gene. The targeted cell lines may then be injected into a mouse blastula stage embryo to generate chimeric founder mice. Heterozygous offspring may be interbred to homozygosity.
As the slowly progressive hearing loss observed in DFNA2 may require the expression from one allele of a dominant negative mutant, it may also be desired to create a knock-in animal in which the wild-type KCNQ4 gene is replaced by this mutated gene.
Animal models for over-expression may be generated by integrating one or more polynucleotide sequence of the invention into the genome according to standard techniques.
The procedures disclosed herein involving the molecular manipulation of nucleic acids are known to those skilled in the art, and are described by e.g. Fredrick M A et al. [Fredrick M A et al.: Short Protocols in Molecular Biology; John Wiley and Sons, 1995] and Sambrook et al. [Sambrook et al.: Molecular Cloning: A Laboratory Manual; 2. Ed., Cold Spring Harbor Lab.; Cold Spring Harbor, N.Y. 1989], and in Alexandra L J (Ed.): Gene Targeting: A practical approach; Oxford University Press (Oxford, New York, Tokyo), 1993.